Protein Unfolding in Freeze Frames: Intermediate States are Revealed by Variable-Temperature Ion Mobility–Mass Spectrometry

The gas phase is an idealized laboratory for the study of protein structure, from which it is possible to examine stable and transient forms of mass-selected ions in the absence of bulk solvent. With ion mobility–mass spectrometry (IM-MS) apparatus built to operate at both cryogenic and elevated temperatures, we have examined conformational transitions that occur to the monomeric proteins: ubiquitin, lysozyme, and α-synuclein as a function of temperature and in source activation. We rationalize the experimental observations with a temperature-dependent framework model and comparison to known conformers. Data from ubiquitin show unfolding transitions that proceed through diverse and highly elongated intermediate states, which converge to more compact structures. These findings contrast with data obtained from lysozyme—a protein where (un)-folding plasticity is restricted by four disulfide linkages, although this is alleviated in its reduced form. For structured proteins, collision activation of the protein ions in-source enables subsequent “freezing” or thermal annealing of unfolding intermediates, whereas disordered proteins restructure substantially at 250 K even without activation, indicating that cold denaturation can occur without solvent. These data are presented in the context of a toy model framework that describes the relative occupancy of the available conformational space.


Theory
A diffusion collision cross section Ω d (T eff ) is a fundamental quantity of a two temperature kinetic theory. 1 Throughout this report, we refer to this quantity as a collision cross section (CCS) which is a so-called fundamental ion mobility relation. [1][2][3][4][5] The expression relating ion mobility (K) to diffusion collision cross section is presented below: Where q -ion charge, N -gas number density, μ -reduced mass of ion and gas molecules, k B -Boltzmann constant. The effective ion temperature (T eff ) is defined as: (2) = + ( ) 2 3 Where T -gas temperature and Eapplied electric field.
In the linear field drift tube ion mobility measurements, we determine the drift times (t d ) -the time ions require to traverse through the drift cell. Particularly in the case of using MS detection, measured time (t measured ) inevitably contains a so-called "dead time" -which corresponds to the time ions spend between the end of the drift region and the detector (t measured = t d + t dead ). Having the times measured across a range of pressure to voltage ratios (P/V), we can determine the mobility from the resulting linear relationship where the slope is equal to 1/K and the dead time corresponds to the intercept.
The resolving power (R) is defined as a ratio of a drift time (t d ) to peaks' full width at half maximum (Δt d ).
For a drift tube ion mobility separator, it depends primarily on diffusion and can be estimated from equation 3: Where L denotes the path length.

Experimental
Experiments were performed on a home-built ion mobility drift tube coupled to a commercial MS platform (QToF2, Micromass, Manchester, UK) as described elsewhere. 6 50 μM solutions were electrosprayed from in-house manufactured nano-ESI tips (P-97, Sutter Instruments, Novato, USA) with the application of 1-1.5 kV. Ions were directed into a Z-spray ion source (Micromass, Manchester, UK) and subsequently into the VT IMS device. Ions were stored for 16 ms prior to release into the drift region.
Pressure at the drift region was held at ~2.1 Torr (Helium). Pressure of the drift gas was monitored with a capacitance manometer (MKS Baratron). Temperature was varied by a combination of liquid nitrogen flow and resistive heating. The temperature was monitored with two platinum resistance (Pt100) thermometers immersed in the drift gas bath. In order to accurately measure the mobility of ions in helium buffer gas, arrival times were measured at several electric fields (3-14 Td) and the linear correlation (R 2 > 0.999) of arrival time vs P/V was obtained, where the slope is inversely proportional to the mobility K and the intercept provides the time spent in the QToF prior to detection (dead time). 7,8 A typical error in CCS due to uncertainty in measurements of pressure, temperature and the P/V  1/K fitting is < 3%.

Framework
The Framework model allows the prediction of the upper and lower CCS boundaries for proteins from their amino acid sequence. Full details of the calculations are described elsewhere. 9,10 In brief, the lower boundary is calculated by assuming the protein in its most folded, globular state occupies a spherical shape. The equation for a protein sphere is as follows where r is the radius of a sphere: Equation 1 The answer from Equation 1 is multiplied by a helium scaling factor of 1.19 to determine the smallest possible CCS value.
The upper boundary assumes the protein amino acid sequence is 'stretched out' so that the alpha carbons in the protein chain are as far apart as theoretically possible. This stretched out protein can be approximated as a cylinder. The equation for the upper boundary is as follows where r is the radius of a cylinder and l is the length of a cylinder: (Å 2 ) = ( 4 ) + 2 2

Equation 2
The answer from Equation 2 is multiplied by a helium scaling factor of 1.19 to determine the largest possible CCS value for a particular protein.
In order to predict low temperature framework values, we have multiplied the answers from Equations 1 and 2 by a factor of 1.07. We know from experimental work shown here and in other VT-IMS work, 6,11 on decreasing the temperature to deep-freeze temperatures (~150-180 K) the CCS increases by approximately 7 %, due to an increased effect of long range ion-molecule interactions at lower temperatures. PSA calculations at lower temperatures also predict an increase in CCS of 5% for lysozyme (295 K compared with 160 K) and 6% for ubiquitin (295 K compared with 150 K), in good agreement with experimental observations. These temperature-scaled framework values are shown as green dashed lines in Figure 3.
We are grateful to Dale Stuchfield for his insights into these calculations.

Figure S8
Mass spectra of disulphide-reduced lysozyme sprayed at a concentration of 30 µM solution 50 mM ammonium acetate and 10mM DTT, pH 6.8. Left: data obtained with no in-source activation at a range of temperatures (160-295 K). Right: the respective data with in-source activation on. Differences in intensity ratio between charge states 7+ and 8+ reflect the variations in day-to-day fine tuning of the n-ESI source.

Figure S13
Mass spectra of alpha-synuclein sprayed at a concentration of 10 µM solution in 100 mM ammonium acetate. Left: data obtained with no in-source activation at a range of temperatures (210-295 K). Right: the respective data with in-source activation on. Differences in intensity ratio between charge states reflect the variations in day-to-day fine tuning of the n-ESI source and ion buncher settings. Figure S14: Summary of experimental CCS distributions for disulfide-reduced lysozyme 7 and 8+ for both non-activated (grey violin plots) and activated (red violin plots).

Figure S15
Hypothetical 1D gas-phase folding free energy surfaces of ubiquitin and lysozyme in the gas phase and in solution.